Lic.-avh. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

B-lymphocytes have traditionally been thought to contribute to immunity and autoimmune disease through terminal differentiation into plasma cells that secrete antibody. However, studies in mice and recent clinical studies have demonstrated that genetically altered B-cell function and B-cell-targeted therapies can significantly affect autoimmune diseases that were predominantly thought to be T-cell-mediated. B-cell depletion in mouse models of disease has also led to the identification of alternative B-cell effector functions that regulate normal immune responses and autoimmune disease. This review highlights multiple B-cell effector mechanisms, including the promotion of cellular immunity, the negative regulation of immune responses, and the production of pathogenic antibodies. / Dissertation

Thesis advisor: Thomas C. Chiles / Normal B lymphocytes require extrinsic factors to grow and proliferate. Surface receptors (e.g., B-cell antigen receptor, BCR) function, in part, to link growth factors to signal transduction/metabolic pathways and the cell cycle machinery. Accumulating evidence indicates that signal transduction-dependent changes in both glucose energy metabolism and de novo transcription of the D-type cyclin-cdk4/6 pathway are necessary for quiescent B-lymphocytes to enter G1-phase of the cell cycle and grow. B cell growth represents a critical checkpoint for subsequent proliferation and clonal expansion of antigen-specific lymphocytes. On the former, we have shown earlier that acquisition of extracellular glucose and metabolism via the glycolytic pathway is required for conventional splenic B-2 lymphocytes to grow (i.e., increase cell size and mass) in response to antigen challenge; however, the metabolic fate and biological significance of glucose-derived carbons are unknown. Here, we show that in response to BCR ligation, glucose carbon flow is directed into a de novo lipogenic pathway that is regulated, in part, via phosphoinositide-3 kinase (PI-3K)-dependent activation of ATP citrate lyase (ACL), a key rate-limiting enzyme in de novo lipogenesis. Inhibition of ACL results in a loss of B-cell growth and cell viability. Regarding the latter point, the B-1a lymphocyte subset expresses cyclins D2 and D3 that are transiently expressed in a non-overlapping manner, notably cyclin D3 expression immediately precedes the G1/S phase transition, suggesting distinct functions for these D-type cyclins in B-1a lymphocyte G0-to-S phase progression. We show herein that murine B-1a cells deficient in cyclin D3 proliferate normally in response to extracellular stimuli, in part, due to a compensatory sustained up-regulation of cyclin D2. In keeping with this, human diffuse large B-cell lymphoma (DLBCL) represents a malignant clonal expansion of B cells characterized by several subsets, including germinal center (GC) and activated B-cell (ABC) types. Here, we show that the GC-type LY18 human DLBCL exhibits constitutive expression of cyclin D3, but not cyclins D1 and D2. Targeting of cyclin D3-holoenzyme complexes with cell permeable chemical- and peptide-based cdk4 inhibitors results in G1-phase arrest and apoptosis via a pathway that involves inhibition of pRb phosphorylation. By contrast, endogenous knock down of cyclin D3 with siRNA did not induce growth arrest or apoptosis, in part, due to redundancy with cyclin E. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A resposta imune ao P. chabaudi se caracteriza pela ativação policlonal de linfócitos B na fase aguda da doença, que resulta em uma intensa produção de anticorpos IgM e IgG de baixa afinidade e autoanticorpos. Apesar de esses anticorpos contribuírem para o controle da infecção, pode retardar a geração de anticorpos de alta afinidade que garantem a destruição dos parasitas e a proteção contra infecções subseqüentes. O nosso objetivo foi caracterizar os mecanismos envolvidos na ativação policlonal dos linfócitos B na infecção pelo P. chabaudi AS. Os nossos resultados mostraram que a cooperação T-B e importante nessa fase da infecção. Os ensaios in vitro mostraram que os linfócitos T da fase aguda da infecção são capazes de estimular linfócitos B naives, aumentando a expressão do CD69 e induzindo proliferação e produção de anticorpos. A molécula de classe II do MHC é importante na ativação policlonal dos linfócitos B, aumentando a expressão do CD69 e induzindo a proliferação de anticorpos, devido ao seu envolvimento na ativação dos linfócitos T. Assim, moléculas como CD28, CD40L e ICOS são importantes na ativação, proliferação e produção de anticorpos. / Polyclonal B cell activation is a feature of the early spleen B cell response to human and murine malaria, which results in intense production of parasite low-affinity IgM and IgG and autoantibodies. Although this response may contribute for the initial parasite control, it may also hinder the generation of high-affinity IgG that guarantees parasite elimination and protection against secondary infections. To characterize the mechanism involved in the polyclonal B cell response to P. chabaudi AS, we have analyzed in vivo and in vitro the role of several molecules known to be involved in antibody production. According to our in vivo results, this response depends on the cooperation of CD4+ T cells with B cells. The in vitro assays shows that the expression of MHC class II molecules is crucial for B cell activation in terms of CD69 expression, proliferation and antibody production, apparently due to their requirement for T cell activation. Signaling pathways mediated by the co-stimulatory receptor CD28, CD40L and ICOS has a central role for B cell proliferative response and antibody production. This study may help to clarify the molecular basis of polyclonal B cell activation induced during acute malaria.

Introdução: A Imunodeficiência Comum Variável (ICV) é a imunodeficiência primária mais prevalente na prática clínica. Suas manifestações vão desde infecções de repetição (que são as mais prevalentes) à neoplasias e doenças autoimunes. Admite-se que o quadro clínico da ICV esteja associado a alterações na diferenciação de linfócitos B (LB), mas também em linfócitos T, monócitos e células dendríticas, além de mutações em genes relacionados a interações entre linfócitos T e B ou homeostasia de linfócitos B. Isto determina uma grande complexidade na classificação destes pacientes, sendo a correlação entre características clínicas e fenotípicas essencial para o melhor entendimento da doença e acompanhamento dos pacientes. Objetivo: Descrever o fenótipo clínico e das subpopulações de LB da coorte de pacientes com ICV acompanhados no Ambulatório de Imunodeficiências Primárias do Hospital das Clínicas e verificar o modelo de classificação que melhor se adequa a estes pacientes. Materiais e Métodos: Após a separação de PBMC e imunofenotipagem dos subtipos de LB de amostras de 70 pacientes, estes foram classificados segundo os critérios das classificações de Freiburgo, Paris e EUROclass. Também foram realizadas a análise das manifestações clínicas; a correlação entre as células de memória e as concentrações séricas das imunoglobulinas e a correlação entre os subtipos de LB e as principais manifestações clínicas desta coorte. Resultados: Esta coorte apresentou um grande distúrbio na distribuição das subpopulações de LB, sendo que todos os pacientes apresentaram redução na frequência de LB de memória com troca de isotipo, mas apenas as pacientes mulheres apresentaram aumento estatisticamente significante nas frequências de LB naive e LB CD21low e redução na frequência de plasmoblastos. Os subtipos de LB transitório e LB de zona marginal não apresentaram diferenças significativas entre pacientes e controles. Não observamos correlação entre as células B de memória e os níveis séricos de imunoglobulinas. A análise clínica revelou diferenças na prevalência de algumas complicações clínicas, entretanto, a correlação entre os subtipos de LB e estas manifestações mostrou associação apenas entre linfadenopatia e redução de plasmoblastos. Por fim, a classificação quanto aos três modelos propostos, revelou que nenhum deles se adequou para esta coorte de pacientes brasileiros. Conclusão: verificamos uma diversidade tanto nas subpopulações de LB quanto na prevalência de algumas manifestações clínicas e por conta destas alterações, até o momento nenhuma classificação se mostrou apropriada para esta coorte. Estes achados reforçam o conceito de heterogeneidade da doença e confirmam a necessidade de um novo modelo de classificação que compreenda outros marcadores. / Introduction: Common Variable Immunodeficiency (CVID) is the primary antibody deficiency most often seen in clinical practice. Patients present different clinical features ranging from recurrent infections (most prevalent) to malignancies and autoimmune disorders. It is accepted that this clinical picture is associated with alterations in the differentiation of B cells but also in T cells, monocytes and dendritic cells, besides mutations in genes related to interactions between T and B cells or in B cell homeostasis. This makes the classification of CVID patients very difficult, on the other hand the association between clinical and immunological phenotype is essential for better understanding of the disease and follow-up of patients. Objective: To describe the clinical and immunological phenotype of a Brazilian cohort of CVID patients followed at the Division of Clinical Immunology and Allergy of University of São Paulo Medical School and to classify them and analyze which is the most appropriated classification for this cohort. Methods and Results: Seventy patients were studied and flow cytometric B cell phenotyping was performed in PBMCs, using a mixture of monoclonal antibodies. The samples were classified according to the Freiburg, Paris and EUROclass classifications. We also analyzed the major clinical manifestations and correlated them to B cell phenotypes, besides correlating memory B cell frequency and serum IgA, IgG and IgM levels. Comparing patients and controls, we found a disturbed frequency of B cell subtypes in this cohort. Both male and female patients presented a reduction on switched memory B cells. However, only female patients had a significant increase on naïve B cells, CD21low B cells and reduction of plasmablasts. Marginal zone and transitory B cells showed no statistical differences between groups. There was no correlation between memory B cell frequency and serum immunoglobulin levels. Clinical analysis revealed a disturbed prevalence in some features but the correlation between B cell subtypes and clinical manifestations revealed only an association between lymphadenopathy and plasmablast reduction. Finally, the classification of this cohort according to the three proposed models revealed that none of them were appropriated to this Brazilian cohort. Conclusion: We observed a diversity in B cell subtypes frequency and the profile of some clinical complications. Because of these findings in both clinical and B cell subtypes, until now no proposed classification is appropriated for this cohort. These findings reinforce the concept of CVID heterogeneity and confirm the necessity of a new classification model that involves other markers

A sepse é a Síndrome da Resposta Inflamatória Sistêmica decorrente de uma infecção por gram positivos/negativos, fungos ou vírus. É caracterizada por alta liberação de mediadores inflamatórios podendo levar à morte. As células B-1 são encontradas em cavidades peritoneal e pleural de camundongos e sua origem e função ainda não são completamente conhecidas. Apresentam marcadores de superfície de linhagem mielóide e linfóide e migram para focos inflamatórios comportando-se como macrófagos. Objetivo: investigar o papel da célula B-1 na resposta inflamatória após estímulo com lipopolissacáride (LPS) in vitro e in vivo. Métodos: TNF-, IL-6, IL-10 (ELISA) e nitrito (Griess) foram dosados em sobrenadante de cultura celular (106 cel./ml). As células em cultura receberam por 24h de estímulo com 10 g/mL de LPS de Escherichia coli (026:B6 Sigma®). Foram realizados os seguintes grupos cultura de célula B-1 (Balb/c), cultura de macrófagos de linhagem (RAW 264.7) coculturas (macrófagos de linhagem RAW 264.7 e células B-1 (Balb/c, C57BL/6 e C57BL/6 IL-10 -/-), e células peritoneais de camundongos Balb/c e Balb/Xid (imunodeficiente em célula B-1) A endotoxemia foi induzida com injeção de LPS 15 mg/kg (i.p.) em camundongos Balb/c e Balb/Xid. Foram quantificados, TNF-, IL-6, IL-10 e nitrito em soro, pulmão e intestino dos animais após 1,5, 4 e 6 horas após a injeção de LPS. Ensaios de inoculação de células B-1 (Balb/c) em camundongos Balb/Xid foram realizados, e curva de sobrevida foi analisada após indução de endotoxemia. Resultados: Após o estímulo com LPS, células B-1 produziram IL-10 e a presença destas células em cocultura com macrófago promoveu a diminuição na produção de TNF-, IL-6, Nitrito e aumento de IL-10. Contudo, célula B-1 (IL-10 -/-) em cocultura com macrófagos, não inibem a produção de mediadores pro inflamatórios. Análise com macrófagos peritoneais de camundongo Balb/Xid e Balb/c após estímulo com LPS em cultura mostrou reprodução do fenômeno encontrado com os experimentos com cultura de célula imortalizada, isto é, maior produção de TNF-, IL-6 e NO em Balb/Xid (B-1 deficiente). Os estudos in vivo mostraram 60% de mortalidade em camundongo Balb/Xid comparando com Balb/c (0%) após 16 horas de injeção de LPS. Nos animais Balb/Xid encontramos padrão pro inflamatório exacerbado com maiores concentrações de TNF-, IL-6 e menores concentrações de IL-10 no plasma e tecidos quando comparamos com Balb/c. Conclusões: Nossos dados mostraram que a presença de células B-1 promoveram diminuição de mediadores pro inflamatórios e aumento de IL-10 em coculturas com macrófagos e que a modulação da resposta inflamatória pode ser devida a secreção de IL-10 pela célula B-1. Este padrão de resposta pro inflamatória se repete in vivo e é a possível causadora da maior taxa de mortalidade em camundongos da linhagem Balb/Xid. / Sepsis syndrome is caused by inappropriate immune activation due to bacteria and bacterial components released during infection. This syndrome is the leading cause of death in intensive care units. Specialized B-lymphocytes located in the peritoneal and pleural cavities are known as B-1 cells. These cells produce IgM and IL-10, both of which are potent regulators of cell-mediated immunity. It has been suggested that B-1 cells modulate the systemic inflammatory response in sepsis. In this study, we conducted in vitro and in vivo experiments in order to investigate a putative role of B-1 cells in a murine model of LPS-induced sepsis. Macrophages and B-1 cells were studied in monocultures and in co-cultures. The B-1 cells produced the anti-inflammatory cytokine IL-10 in response to LPS. In the B-1 cell-macrophage co-cultures, production of proinflammatory mediators (TNF-, IL-6 and nitrite) was lower than in the macrophage monocultures, whereas that of IL-10 was higher in the co-cultures. Co-culture of B-1 IL-10/ cells and macrophages did not reduce the production of the proinflammatory mediators (TNF-, IL-6 and nitrite). After LPS injection, the mortality rate was higher among Balb/Xid mice, which are B-1 cell deficient, than among wild-type mice (65.0% vs. 0.0%). The Balb/Xid mice also presented a proinflammatory profile of TNF-, IL-6 and nitrite, as well as lower levels of IL-10. In the early phase of LPS stimulation, B-1 cells modulate the macrophage inflammatory response, and the main molecular pathway of that modulation is based on IL-10-mediated intracellular signaling.

A sepse é causada por microorganismos tais como: bactérias Gram-negativas, Gram-positivas, fungos e vírus. A sepse grave e o choque séptico estão associados a taxas de mortalidade de 40 a 60%. O evento mais importante para a evolução do quadro séptico é a apoptose das células efetoras do sistema imune. A eliminação de um grande número de células do sistema imune compromete a defesa efetiva do hospedeiro. Para estudarmos o papel das células imunes na sepse utilizamos o modelo de tolerância ao LPS. Em nossos estudos induzimos tolerância ao LPS em camundongos Balb-C e analisamos os padrões da expressão gênica nos linfócitos do baço. Para o mapeamento dos genes, utilizamos o macroarray, identificamos o grupo funcional de genes que tem maior relevância na proteção induzida pela tolerância, e em seguida confirmamos os resultados encontrados através do RT-PCR, Western Blotting, atividade de caspase 1 e citometria de fluxo. Encontramos uma importante redução na expressão de genes, como heat shock proteins, óxido nítrico e apoptose. Após análise da membrana contendo os genes integradores da resposta produzida pela tolerância, optamos por enfatizar inicialmente os genes envolvidos no processo apoptótico devido à relevância deste processo no quadro séptico conforme mostraram os trabalhos encontrados na literatura. Demonstramos que animais tolerantes ao LPS apresentam diminuição dos eventos apoptóticos, com redução na expressão dos genes ligados às caspases 7, 8 e 11, assim como a redução dos genes ligados ao Bid e Apaf-1. Ao analisarmos o RNAm através do RT-PCR, encontramos redução na Caspase 3, Bax e Bcl2, resultado que se confirmou ao analisarmos a expressão protéica através do Western- Blotting. Realizamos citometria de fluxo para avaliarmos a ocerrência de apoptose nos linfócitos dos animais submetidos à ligadura cecal. Confirmamos uma redução da apoptose e necrose em linfócitos dos animais tolerantes em relação aos controles. Com estes resultados podemos propor que a tolerância seria benéfica na redução da apoptose e controle do quadro séptico, além disso, a diminuição na expressão do gene ligado à caspase 11 estaria contribuindo para a diminuição do quadro inflamatório, pois a caspase 11 é um mediador essencial na resposta ao choque séptico. / Sepses are caused by microorganisms such as: Gram-negative bacteria, Gram-positive bacteria, fungus and virus. Several sepses and the septic shock have been associated with the mortality rates from 40 to 60%. One of the most important events for the septic evolution is the apoptosis cells of the immune system. The cells immune system elimination compromises the host. We induced LPS tolerance in Balb-C mice and analyzed genes expressions in spleens; we found an important reduction in the genes expressions like: heat shock proteins, nitric oxide and apoptosis. After analysis of the membrane containing the sepses genes produced by tolerance, we emphasized initially the genes involved in the apoptosis. Demonstrated that animals LPS tolerants presented decrease in apoptotic events, with reduction in the genes expression related caspases 7, 8, 11, Bid and Apaf-1. In the mRNA by RT-PCR, we found a reduction in Caspase 3, Bax and Bcl2, and these results were confirmed by Western-blot. We realized flow cytometry and we showed that the results are maintained, presenting reduction in both the apoptotic and necrotic events in tolerants animals. These results showed that the tolerance would be favorable in the apoptosis reduction and control of the sepsis, moreover, the expression reduction to caspase 11 genes would be contributing to the inflammatory reduction because Caspase 11 is an essential mediator in the septic shock.

Human T cells may be genetically modified to express targeted chimeric antigen receptors (CARs). We have previously demonstrated that T cells modified to express a CAR specific to the B cell tumor antigen CD19, termed 19-28z, successfully eradicate systemic human CD19+ tumors in SCID-Beige mice. While these results are encouraging, this xenogeneic tumor model fails to address potential limitations of this therapeutic approach in the clinical setting wherein these modified T cells encounter a hostile tumor microenvironment. Specifically, these models fail to address potential effector T cell inhibition mediated by endogenous regulatory T cells (Tregs). To investigate the role of inhibitory Tregs, we initially assessed the in vitro function of CAR-modified T cells in the context of Tregs. We found that CD19-targeted T cell proliferation and cytotoxicity were inhibited by purified natural Tregs. To further assess the role of these Tregs in vivo, we isolated and genetically modified Tregs to express the CD19-targeted 19z1 CAR. We verified specific trafficking of targeted Tregs to CD19+ tumors in vivo, and demonstrate that 19z1 Tregs wholly inhibit anti-tumor function of subsequently injected 19-28z effector T cells even at low Treg to effector T cell ratios (1:8). In order to overcome this limitation, we assessed whether the addition of a pro-inflammatory cytokine in vitro could overcome Treg inhibition. Indeed, the addition of exogenous IL-12 mediated resistance of 19-28z T cells to Treg inhibition. In light of this data we generated a bicistronic retroviral vector containing both the 19-28z CAR as well as the murine IL-12 fusion gene (19-28z IRES IL-12). Significantly, we found that 19-28z/IL-12+ T cells when compared to 19-28z+ T cells exhibited enhanced proliferation in vitro as well as resistance to Treg mediated inhibition. Finally, we demonstrate that 19-28z/IL-12+ T cells overcome Treg inhibition in vivo in our SCID-Beige Treg tumor model. In conclusion, tumor targeted T cells modified to express IL-12 demonstrate significantly enhanced in vivo anti-tumor efficacy in the presence of Tregs that are similarly targeted to the site of tumor. These results validate utilization of IL-12 secreting tumor targeted T cells in future clinical trials.

The location of the immunologic response to an allograft is not known with certainty. However, organized collections of T cells, B cells and antigen presenting cells have been found in peripheral tissue, in close proximity to organs undergoing rejection. It is hypothesized that this tertiary lymphoid tissue may be a location in which activation of lymphocytes can occur, leading to rejection of an allograft. We report here that in a splenectomized aly/aly mouse, which is devoid of secondary lymphoid organs and will normally fail to reject an allograft, the presence of tertiary lymphoid organs is associated with graft rejection. We additionally find that tertiary lymphoid organs can act as lymph nodes, and can support effector and memory allograft rejection responses. It is demonstrated that ectopic lymphoid tissue in aly/aly mice will support the multiplication and transformation of transferred naïve CD4 and CD8 T cells into cells that display phenotypic markers characteristic of effector and memory lymphocytes. These results demonstrate that ectopic lymphoid tissue is associated with the loss of immunologic ignorance and is sufficient to enable graft rejection. This suggests that allograft rejection may take place within ectopic lymphoid tissue, and suggests that techniques to interfere with the development of this tissue might offer a therapeutic approach to preserving organ allografts.